Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

Patterns of genome size variation in caridean shrimps: new estimates for non-gambarelloides Synalpheus species.

Genome size (GS) or DNA nuclear content is considered a useful index for making inferences about evolutionary models and life history in animals, including taxonomic, biogeographical, and ecological scenarios. However, patterns of GS variation and their causes in crustaceans are still poorly understood. This study aimed to describe the GS of five Neotropical Synalpheus non-gambarelloides shrimps (S. apioceros, S. minus, S. brevicarpus, S. fritzmueller, and S. scaphoceris) and compare the C-values of all Caridea infraorder in terms of geography and phylogenetics. All animals were sampled in the coast of São Paulo State, Brazil, and GS was assessed by flow cytometry analysis (FCA). The C-values ranged from 7.89 pg in S. apioceros to 12.24 pg in S. scaphoceris. Caridean shrimps had higher GS than other Decapoda crustaceans. The results reveal a tendency of obtaining larger genomes in species with direct development in Synalpheus shrimps. In addition, a tendency of positive biogeographical (latitudinal) correlation with Caridea infraorder was also observed. This study provides remarkable and new protocol for FCA (using gating strategy for the analysis), which led to the discovery of new information regarding GS of caridean shrimps, especially for Neotropical Synalpheus, which represents the second-largest group in the Caridea infraorder.

Network Analysis of Inflammatory Bowel Disease Research: Towards the Interactome.

BACKGROUND AND AIMS: Modern views accept that inflammatory bowel diseases [IBD] emerge from complex interactions among the multiple components of a biological network known as the 'IBD interactome'. These diverse components belong to different functional levels including cells, molecules, genes and biological processes. This diversity can make it difficult to integrate available empirical information from human patients into a collective view of aetiopathogenesis, a necessary step to understand the interactome. Herein, we quantitatively analyse how the representativeness of components involved in human IBD and their relationships ha ve changed over time. METHODS: A bibliographic search in PubMed retrieved 25 971 abstracts of experimental studies on IBD in humans, published between 1990 and 2020. Abstracts were scanned automatically for 1218 IBD interactome components proposed in recent reviews. The resulting databases are freely available and were visualized as networks indicating the frequency at which different components are referenced together within each abstract. RESULTS: As expected, over time there was an increase in components added to the IBD network and heightened connectivity within and across functional levels. However, certain components were consistently studied together, forming preserved motifs in the networks. These overrepresented and highly linked components reflect main 'hypotheses' in IBD research in humans. Interestingly, 82% of the components cited in reviews were absent or showed low frequency, suggesting that many aspects of the proposed IBD interactome still have weak experimental support in humans. CONCLUSIONS: A reductionist and fragmented approach to the study of IBD has prevailed in previous decades, highlighting the importance of transitioning towards a more integrated interactome framework.

Karyotype description and comparative chromosomal mapping of rDNA and U2 snDNA sequences in Eigenmannialimbata and E.microstoma (Teleostei, Gymnotiformes, Sternopygidae).

The genus Eigenmannia Jordan et Evermann,1896 includes electric fishes endemic to the Neotropical region with extensive karyotype variability and occurrence of different sex chromosome systems, however, cytogenetic studies within this group are restricted to few species. Here, we describe the karyotypes of Eigenmannialimbata (Schreiner et Miranda Ribeiro, 1903) and E.microstoma (Reinhardt, 1852) and the chromosomal locations of 5S and 18S rDNAs (ribosomal RNA genes) and U2 snDNA (small nuclear RNA gene). Among them, 18S rDNA sites were situated in only one chromosomal pair in both species, and co-localized with 5S rDNA in E.microstoma. On the other hand, 5S rDNA and U2 snRNA sites were observed on several chromosomes, with variation in the number of sites between species under study. These two repetitive DNAs were observed co-localized in one chromosomal pair in E.limbata and in four pairs in E.microstoma. Our study shows a new case of association of these two types of repetitive DNA in the genome of Gymnotiformes.

Meiotic self-pairing of the Psalidodon (Characiformes, Characidae) iso-B chromosome: A successful perpetuation mechanism.

B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)15 microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.

Cytogenetics from the Dock: A New Postmortem Protocol Makes It Possible to Obtain Cytogenetic Preparations from Marine Fish.

Cytogenetic studies in marine fish are scarce, and elemental cytogenetic information is available for not >2% of the species. Traditional cytogenetic methods require living individuals for their application, making the analysis of marine ichthyofauna very difficult. In this study, we present a detailed new protocol to obtain cytogenetic preparations from marine fish, through access to specimens in postmortem condition. The application of this protocol made it possible to access elemental cytogenetic information (diploid number) in six native species of the South Pacific Ocean, representative of five orders. In this way, we provide a new low-cost methodological tool for focused or large-scale cytogenetic analysis, both in economically important, native, or threatened species.

Cytogenetic characterization, rDNA mapping and quantification of the nuclear DNA content in Seriolella violacea Guichenot, 1848 (Perciformes, Centrolophidae).

Seriolella violacea Guichenot, 1848 is an important component of the fish fauna of the Chilean coast and is of great economic interest. Cytogenetic information for the family Centrolophidae is lacking and the genomic size of five of the twenty-eight species described for this family are is barely known. This study aimed to describe for the first time the karyotype structure via classical and molecular cytogenetics analysis with the goal of identifying the constitutive heterochromatin distribution, chromosome organization of rDNA sequences and quantification of nuclear DNA content. The karyotype of S. violacea is composed of 48 chromosomes, with the presence of conspicuous blocks of heterochromatin on chromosomal pairs one and two. FISH assay with a 5S rDNA probe, revealed the presence of fluorescent markings on the heterochromatic block of pair one. The 18S rDNA sites are located exclusively on pair two, characterizing this pair as the carrier of the NOR. Finally, the genomic size of S. violacea was estimated at 0.59 pg of DNA as C-value. This work represents the first effort to document the karyotype structure and physical organization of the rDNA sequences in the Seriolella genome, contributing with new information to improve our understanding of chromosomal evolution and genomic organization in marine perciforms.

Modulating offspring responses: concerted effects of stress and immunogenic challenge in the parental generation.

The perception, processing and response to environmental challenges involves the activation of the immuno-neuroendocrine (INE) interplay. Concerted environmental challenges might induce trade-off when resource allocation to one trait occurs at the expense of another, also producing potential transgenerational effects in the offspring. We evaluated whether concerted challenges, in the form of an immune inoculum against inactivated Salmonella enteritidis (immune challenge, ICH) and a chronic heat stress (CHS) exposure on adult Japanese quail, modulate the INE responses of the parental generation and their offspring. Adults were inoculated and later exposed to a CHS along nine consecutive days. For the last 5 days of the CHS, eggs were collected for incubation. Chicks were identified according to their parental treatments and remained undisturbed. Induced inflammatory response, heterophil/lymphocyte (H/L) ratio and specific humoral response against sheep red blood cells (SRBC) were evaluated in both generations. Regardless of the ICH, stressed adults showed a reduced inflammatory response and an elevated H/L ratio compared with controls. In offspring, the inflammatory response was elevated and the specific SRBC antibody titres were diminished in those chicks prenatally exposed to CHS, regardless of the ICH. No differences were found in the H/L ratio of the offspring. Together, our results suggest that CHS exposure influences the INE interplay of adult quail, establishing trade-offs within their immune system. Moreover, CHS not only affected parental INE responses but also modulated their offspring INE responses, probably affecting their potential to respond to future challenges. The adaptability of the developmental programming of offspring would depend on the environment encountered.

Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes.

The origin of supernumerary (B) chromosomes is clearly conditioned by their ancestry from the standard (A) chromosomes. Sequence similarity between A and B chromosomes is thus crucial to determine B chromosome origin. For this purpose, we compare here the DNA sequences from A and B chromosomes in the characid fish Characidium gomesi using two main approaches. First, we found 59 satellite DNA (satDNA) families constituting the satellitome of this species and performed FISH analysis for 18 of them. This showed the presence of six satDNAs on the B chromosome: one shared with sex chromosomes and autosomes, two shared with sex chromosomes, one shared with autosomes and two being B-specific. This indicated that B chromosomes most likely arose from the sex chromosomes. Our second approach consisted of the analysis of five repetitive DNA families: 18S and 5S ribosomal DNA (rDNA), the H3 histone gene, U2 snDNA and the most abundant satDNA (CgoSat01-184) on DNA obtained from microdissected B chromosomes and from B-lacking genomes. PCR and sequence analysis of these repetitive sequences was successful for three of them (5S rDNA, H3 histone gene and CgoSat01-184), and sequence comparison revealed that DNA sequences obtained from the B chromosomes displayed higher identity with C. gomesi genomic DNA than with those obtained from other Characidium species. Taken together, our results support the intraspecific origin of B chromosomes in C. gomesi and point to sex chromosomes as B chromosome ancestors, which raises interesting prospects for future joint research on the genetic content of sex and B chromosomes in this species.

Genetic and Chromosomal Differentiation of Rhamdia quelen (Siluriformes, Heptapteridae) Revealed by Repetitive Molecular Markers and DNA Barcoding.

Rhamdia quelen, a species of Heptapteridae, is considered to be a complex because of taxonomic and phylogenetic inconsistencies. Determining the physical location of repetitive DNA sequences on the chromosomes and the DNA barcode might increase our understanding of these inconsistencies within different groups of fish. To this end, we analyzed R. quelen populations from two river basins in Brazil, Paraguay and Parana, using DNA barcoding and different chromosomal markers, including U2 snDNA, which has never been analyzed for any Rhamdia species. Cytochrome c oxidase I gene sequence analysis revealed a significant differentiation among populations from the Miranda and Quexada rivers, with genetic distances compatible to those found among different species in neotropical fishes. Our results, in general, revealed a conservative chromosomal evolution in R. quelen and a differential distribution of some markers, such as 5S rDNA and U2 snDNA, in different populations. We suggest that R. quelen must undergo a major revision in its morphological, genetic, and cytogenetic molecular and taxonomic structure to elucidate possible operational taxonomic units.

Chromosomal organization of four classes of repetitive DNA sequences in killifish Orestias ascotanensis Parenti, 1984 (Cyprinodontiformes, Cyprinodontidae).

Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems.

ZZ/Z0: The New System of Sex Chromosomes in Eigenmannia aff. trilineata (Teleostei: Gymnotiformes: Sternopygidae) Characterized by Molecular Cytogenetics and DNA Barcoding.

The cytogenetic characteristics of Eigenmannia aff. trilineata were analyzed by basic and molecular cytogenetics, applying fluorescent in situ hybridization, with 18S and 5S rDNA and U2 snRNA probes. The species revealed a kind of polymorphism associated to ZZ/Z0 type sex chromosomes, with 2n = 32 (8m+2sm+22a, NF = 42) in all males under analysis, whereas females evidenced 2n = 31 (8m+1sm+22a, NF = 40). C-banding showed constitutive heterochromatin restricted to the pericentromeric region of all chromosomes and single-nucleolus organized regions on pair 11. A site for rDNA 5S was synthetic with a cluster of rDNA 18S near the centromere on the long arm of only one homologue of pair 11. Other clusters for 5S rDNA were sited on pairs 7, 10, 12, 13, and 16. Further, 5S rDNA was co-located with U2 cluster in the pericentromeric region of pair 12. Joint analysis of DNA barcoding from cytochrome c oxidase subunit I (COI) sequences, generated from the karyotyped samples of E. aff. trilineata, and sequences of other Gymnotiforms recognized E. aff. trilineata as an Operational Taxonomic Unit. Results foreground the hypothesis that cytotypes are independent evolution units as cryptic species with a low morphological differentiation level, although with high genetic/karyotype differentiation rates.

Chromosomal Mapping of Repetitive DNA Sequences in the Genus Bryconamericus (Characidae) and DNA Barcoding to Differentiate Populations.

The mapping of repetitive DNA sites by fluorescence in situ hybridization has been widely used for karyotype studies in different species of fish, especially when dealing with related species or even genera presenting high chromosome variability. This study analyzed three populations of Bryconamericus, with diploid number preserved, but with different karyotype formulae. Bryconamericus ecai, from the Forquetinha river/RS, presented three new cytotypes, increasing the number of karyotype forms to seven in this population. Other two populations of Bryconamericus sp. from the Vermelho stream/PR and Cambuta river/PR exhibited interpopulation variation. The chromosome mapping of rDNA sites revealed unique markings among the three populations, showing inter- and intrapopulation variability located in the terminal region. The molecular analysis using DNA barcoding complementing the cytogenetic analysis also showed differentiation among the three populations. The U2 small nuclear DNA repetitive sequence exhibited conserved features, being located in the interstitial region of a single chromosome pair. This is the first report on its occurrence in the genus Bryconamericus. Data obtained revealed a karyotype variability already assigned to the genus, along with polymorphism of ribosomal sites, demonstrating that this group of fish can be undergoing a divergent evolutionary process, constituting a substantive model for studies of chromosomal evolution.

Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae).

Characidium gomesi Travasso, 1956 specimens from the Pardo River have up to four heterochromatic supernumerary chromosomes, derived from the sex chromosomes. To access the meiotic behavior and distribution of an active chromatin marker, males and females of Characidium gomesi with two or three B chromosomes were analyzed. Mitotic chromosomes were characterized using C-banding and FISH with B chromosome probes. Meiocytes were subjected to immunofluorescence-FISH assay using anti-SYCP3, anti-H3K4m, and B chromosomes probes. Molecular homology of supernumeraries was confirmed by FISH and by its bivalent conformation in individuals with two of these chromosomes. In individuals with three Bs, these elements formed a bivalent and a univalent. Supernumerary and sex chromosomes exhibited H3K4m signals during pachytene contrasting with their heterochromatic and asynaptic nature, which suggest a more structural role than functional of this histone modification. The implications of this result are discussed in light of the homology, meiotic nuclear organization, and meiotic silencing of unsynapsed chomatin.

Uncovering the Ancestry of B Chromosomes in Moenkhausia sanctaefilomenae (Teleostei, Characidae).

B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tietê rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B1), while the other is darkly C-banded (heterochromatin-like) (B2); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B2-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B2 type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4-6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive.

Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes.

BACKGROUND: Different moderrn methodologies are presently available to analyze meiotic chromosomes. These methods permit investigation of the behavior of chromosomes in the normal complement and of sex and B chromosomes, two special types of chromosomes that are associated with the A complement and are present in many organisms, including fishes. However, meiotic studies are still scarce in fishes, considering the wide number of species in this group.. Here, we describe a new protocol for the visualization of the synaptonemal complex in spermatocytes and oocytes of fishes and to the sequential use of the technique with other procedures and techniques such as immunodetection of the synaptonemal complex protein with a specific antibody and co-detection of DNA sequences by FISH. RESULTS: The meiotic surface-spreading protocol used in the present proposal worked well in representative species of four fish orders and was useful in obtaining good results even in small specimens. Fish-specific antibodies and commercial products worked similarly well to detect synaptonemal complex (SC) proteins. The sequential application of fluorescence in situ hybridization using specific probes showed clear signals associated with the SC structures identified by immunostaining. CONCLUSION: Here, we provide a useful and applicable immunofluorescent protocol for the visualization of synaptonemal complex proteins in the meiotic cells of fishes in surface-spreading preparations. Furthermore, this technique allows for the sequential application of other cytogenetic procedures.

Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes).

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.